KR20000029764A - Active substance screening process - Google Patents

Abstract

PURPOSE: A screening process is provided to test a plurality of samples of an active substance within very short time. CONSTITUTION: An apparatus for active substance screening process includes a scanning nearfieldoptical microscope (SNOM), a device for receiving single or multiple samples and a control unit. An active substance is detected by observing changes in a signal (S) by means of the SNOM.

Description

Screening method of active substance {Active Substance Screening Process}

The present invention relates to methods of screening active compounds, in particular mass screening methods in the field of active compounds. The invention also relates to the use of a near-microscope instrument and to an apparatus comprising a near-microscope instrument and a control device. The invention also relates to nanoscale titration instruments.

New basic structures, ie ligands, agonists or active compounds, which have been improved with respect to the action on enzymes or receptors, DNA nucleases or polymerases, proteases such as thrombin, membrane receptors, nuclear receptors or binding proteins The discovery of such molecular structures is still a laborious and time-consuming process of research to this day.

Its success depends heavily on contingency as it has to scan a very large number of substances until it finds an active substance for a target molecule, for example a receptor. Bioactivity studies of many, for example, 100,000 to 1,000,000 substances, take very long time, considering that activity studies with cells or enzymes require long incubation times. Thus, many of these materials can be tested and investigated quickly only if the tests are equally very sensitive and very fast.

In many cases fluorescence is used for this test. It is very sensitive but also prone to failure. In addition, this test method is very time consuming because it requires multiple additional processing steps from the actual culture to the measurement process. For example, they include a washing step to increase the signal to noise ratio, in particular by reducing the background fluorescence. In general, it can be said that improving the sensitivity of the detection method slows down the test. Simultaneous resolution by either a single test method or a corresponding device for these two problems has so far been impossible.

Recently, the discovery of Scanning Tunneling Microscopy by Binig and Rohrer [G. Binnig, H. Rohrer, C. Gerber, E. Weibel, Phys, Rev. Lett. 49 (1982), 57, have led to rapid advances in the field of ultra-high sensitivity scanning probe methods.

In scanning tunneling microscopes, metal tips are moved (scanned) line by line on conductive or semiconducting samples. The tunneling current generated at a very small distance between the tip and the sample is a control parameter, recording the height profile and / or electronic structure profile of the surface being inspected. It examines the structure at atomic resolution of about 0.1 nm under standard laboratory conditions. In 1986, this was applied to nonconductive samples via scanning atomic force microscopy (SAFM). Binnig, C. F. Quate, C. Gerber, Phys. Rev. Lett. 56 (1986) 930]. This is due to the examination of polymeric materials [G. Kraussch, M. Hipp, M. Boltau, O. Marti, J. Mlynek, Macromolecules 28 (1995) 260] and biological structures [C. Bustamante, D. Keller, Physics Today, December 1995, 32]. Scanning in SAFM is performed using silicon or silicon nitride tips, which are usually pyramidal on a flexible cantilever. The tilt of the cantilever is generally optically measured and used to reconstruct the height profile.

Scanning near-field optical microscopy (SNOM or NSOM) is achieved when an optical fiber to which laser light is supplied moves as a probe on the sample instead of the aforementioned tip, and light reflected or transmitted according to the sample characteristics is detected with the aid of a photodetector [ H. Heinzelmann, D.W. Pohl, Appl. Phys. A 59 (1994) 89; E. Betzig, J. Trautman, Science 257 (1992) 189]. By avoiding the refraction limits that occur in conventional optics, this method using all conventional optical contrast mechanisms such as adsorption, reflection, polarization, fluorescence, etc., allows very high local resolution down to the nanometer range. Sensitivity to detecting individual fluorescent molecules in fluorescence microscopy is achieved [E. Betzig, R.J. Chichester, Science 262 (1993) 1422]. This method is shown schematically in FIG. 1.

In addition to the SNOM devices described above as examples above, additional devices are described in the prior art. For example, tetrahedral tips (Koglin J. et al., Photos and Local Probes.Proc. Of the NATO Advanced Research Workshop.Ed. Mart O. et al.Kluwer Academic Publ. 1995) or, for example, light Conductive tips (Akamine S. et al., Proceedings IEEE Micro Electro Mechanical Systems 1995, page 155) can be used.

This technique has only been used in sporadic investigations in the biological field, for example, S. Smith et al., Proc. SPIE-Int. Soc. Opt. Eng. 1855, 81 (1994) (Scanning Probe Microscopies II) or D.M.N. Jondle et al., Chromosome Research 3, 239 (1995). A second reference describes the investigation of the ultrastructure of polyten chromosomes from salivary glands of fruit flies by SAFM.

It is an object of the present invention to provide a method for screening active compounds which can test a large number of samples in a very short time. This method not only saves time, but requires as little work as possible compared to conventional methods up to now.

The inventors have found that this object can be achieved by a method for detecting active compounds from changes in signal [S] that can be observed by scanning near-field microscopy (SNOM instruments). Accordingly, the present invention relates to a device comprising a use of a SNOM instrument in a screening method and an adjustment mechanism capable of selecting a local microscope instrument, a holder for a single or multiple samples, and an individual compartment of a single or multiple sample holder by the SNOM instrument. will be.

Preferred embodiments of the invention are the subject matter of the dependent claims.

1 shows a basic structure of a SNOM device using an optical fiber.

2a) and b) shows SNOM before [a)] or after [b)] adding a solution comprising active compound W ₂ and additional active compound Wx to a complex of receptor R ₁ and fluorescently-labeled active compound W ₁ . The reaction step which shows the novel screening method using was shown.

Figures 3a) and b) show the same reaction steps as in Figures 2a) and b) where the positions of the receptor R ₁ and the active compound W ₁ are interchanged.

4 shows reaction steps taking place in an embodiment of a novel method in the form of a sandwich technology.

5 shows a further embodiment of the novel method in which the unknown active compound Wx is detected indirectly from structural changes in the receptor.

<Description of the code | symbol about drawing>

1

1: optical fiber

2: sample / substrate

3: light source (eg Ar laser)

4: control unit for optical fiber

5: photodetector

6: detection electronics

2

1: optical fiber

2: substrate (eg, Au deposition coated Si wafer, cellular structure)

R1: receptor

W1: active compound

F: fluorescent label

3

1: optical fiber

2: substrate (eg, Au deposition coated Si wafer, cellular structure)

R1: receptor

W1: active compound

F: fluorescent label

4

Sandwich technology

5

Sandwich technology

The SNOM instrument, as shown schematically in FIG. 1, is for example provided to an optical fiber 1 and a probe 2 which are supplied with Ar-laser light from a light source 3 and carried out on the probe 2 in a control unit 4. In principle, a photodetector 5 with detection electronics 6 for reflected or transmitted light is shown.

As already mentioned above, the present invention also relates to methods of screening in the field of mass screening in which the active compounds, in particular SNOM technology, are used. With the help of this technique, it is possible to detect the fluorescence of individual molecules when the distance between the probe and the sample is in the range of several nm. However, the use of SNOM technology in the novel method is in principle not limited to the field of fluorescence, and can be used for any detection method in which the new active compound to be found causes a change in the optical signal. As a result, the optical signal can be adsorption, polarization or change in wavelength dependent on these properties, independent of fluorescence.

The SNOM technique used in the novel method of the present invention is likewise not limited as shown in FIGS. 1, 2 and 3, and the optical proximity is provided by the optical fiber. All scanning optics can be used as long as they provide near field at similar resolutions where similar resolutions as possible using optical fibers can be achieved. These include, for example, the tetrahedron or photoconductive tip described above.

SNOM can be advantageously used, for example, in the following manner for the screening of active compounds.

First, receptor R1 (FIG. 2) or control active W1 compound (FIG. 3) is immobilized on a suitable surface of the substrate (2). The term "receptor" is used herein to include, for example, fibrinogen, endothelin, DNA or RNA, and in most cases proteins and lipids, but also very broadly, including parts of whole cells or tissues. The immobilized material is also referred to as molecule A below. The substrate may be a gold-coated deposition wafer, a modified, for example, silanized silicon surface or other planar substrate, such as mica, graphite, and the like. It is also possible to use biological substrates, but it may be necessary to pre-fix them on the inorganic substrates described above. The immobilized receptor (R ₁ ) (FIG. 2) or the control active compound (W ₁ ) (FIG. 3) is characterized by the fluorescently-labeled active compound (W ₁ -F) or the fluorescently-labeled receptor (R ₁ -F). Occupied, and itself blocked. When higher affinity active compound W ₂ is added to form stronger bonds [FIG. 2B) or [FIG. 3B)], the control active compound (W ₁ ) is released from the receptor binding site and the active compound W ₂ is Combined. Thus, the fluorescently labeled active compound W ₁ is released from the surface (FIG. 2B). If the control active compound W ₁ is immobilized on the substrate and the two active compounds W ₁ and W ₂ are unfixed, but compete for binding to the fluorescently-labeled receptor, the same reaction occurs in the opposite case (FIG. 3). ). In both cases, the fluorescence recorded by the optical fiber directly above the receptor apparently decreases or even falls to zero. However, it can also only modulate the modulation, for example wavelength dependence. In this case, this is not the intensity, but the color of the changing fluorescence.

A particular advantage of the novel method using SNOM technology is that it does not play a substantial role since fluorescent molecules that move freely in solution by dispersion are not detected by SNOM probes. The volume resulting from the information related to SNOM use is very small because of the high distance dependence of the signal. The volume is from several nm ³ up to some 10,000 nm ³ . Thus, freely dispersed fluorescent molecules play no role in this method and have a very good signal-to-noise ratio. This has the important advantage that the time-consuming post-treatment of the sample by, for example, repeated washing is unnecessary for the very sensitive measurement methods known to date.

In this respect, it is also clear that the short distance provided by the optical fiber is not important in the novel method. Any method of providing a suitable locality has the advantages described above.

Further embodiments of the novel method are shown in FIG. 4. This is different from the method shown in FIGS. 1 to 3 described in that the novel active compound Wx is detected directly. First, the unknown active compound Wx binds to receptor R ₁ . This novel active compound Wx has a domain (*) in which it differs from the known active compound W ₁ . To detect this domain, an antibody labeled with a known molecular probe such as fluorescein isothiocyanate can be used. This fluorescently-labeled antibody binds to a complex of the active compounds Wx and R ₁ . In this method the complex can be detected directly by the detected fluorescence.

Further embodiments of the novel method are shown in FIG. 5. This embodiment differs from that shown in FIG. 4 in that the fluorescently-labeled antibody does not directly bind to the unknown active compound Wx, but changes its structure during binding to the receptor R ₁ (another site steric effect). This change in structure exposes new structural domains on the receptor. Biological molecular probes, such as fluorescently-labeled antibodies, bind these domains. In this way the novel active compound Wx can be detected indirectly via the fluorescence generated.

In the above case, the active compound may be added from a suitable solvent, for example an aqueous or ethanol salt solution or a suitable solvent. The measurement itself can be carried out in a solvent or on a dried sample. The analysis of the active compound can be carried out in the same way for a mixture of active compounds, including mixtures with other compounds, without having to be carried out individually (see FIG. 2). Fixation is possible using an ELISA plate [96-well nano scale titration plate] and a corresponding sample and solution processing system such as an automated pipette for nano scale structural systems. Of course, it is also possible to analyze receptors immobilized on cellular structures in the same way.

The immobilization of a molecule—a receptor, ligand or other molecule, for example a cellular binding protein on a receptor or substrate on a neuroreceptor or immune system cell—can be covalent or noncovalent. Non-covalent fixation occurs, for example, by direct adsorption. However, fixation can also occur via covalent bonds when the substrate has a suitably activated surface. For example, in liquid adsorption the substrate surface (self-assembly [S. Akari et al., Adv. Mater. 7 (1995) 549)) can be activated by the use of thiolcarboxylic acids. Thiol bonds acid groups on the substrate, for example, on the surface of gold, to face the outside by adsorption.

Bonding of molecule A cannot occur directly on the substrate surface or thiolcarboxylic acid. It may also be indirect. For example, a spacer can be arranged between the thiolcarboxylic acid described above and molecule A such as a protein, a receptor, a ligand, and the like. Spacers of this type are aminohexanoic acid or spermidine. Such molecules may bind to the aforementioned thiolcarboxylic acids via amino groups.

Subsequently, molecules, ie receptor active compounds, ligands, and the like, bind to the substrate surface. In the above case, this bonding occurs covalently through the spacer.

If the insertion of the spacer between the substrate surface and molecule A to be analyzed is still inadequate for several reasons, for example due to inadequate conformation, the insertion of the spacer can also be repeated several times.

For example, two or more molecules of the spacer aminohexanoic acid described above may be linked to each other. The only limitation on the number of connected spacers is that if the distance between the surface of the substrate and the molecules A joined via the spacer is too large, very long spacers are naturally correspondingly flexible, thus making the use of SNOM impossible. It literally no longer happens.

Because of the principle of measurement, it is very particularly advantageous that the combination of methods is possible. Instead of a single optical fiber, it is also possible to use multiple fibers simultaneously. The detection of fluorescence can then take place via a fiber coupler and / or detector arrangement. Thus, it is possible to directly decipher a large number of wells, such as ELISA plates or whole plates, generally into 96 detection sites.

However, this is advantageously carried out using a novel apparatus comprising a near field microscope instrument, a single or multiple sample holder and a control mechanism for addressing individual compartments of the single or multiple sample holder by the SNOM instrument. It is of course also possible for the control method to be reversed, ie to address the near-field microscope instrument by a moving multiple sample holder in a similar manner as a moving sample step in a conventional microscope.

Multiple sample holders in this instrument are advantageously the same as those used in multiwell nanoscale titration plates, eg ELISA tests. Multiple sample holders of this type preferably have miniaturized dimensions to increase the speed of the novel active compound screening method. In this case the lower limit on the size (diameter) of the individual compartments is only determined by the size of the optical fiber. Therefore, the lower limit is about 10 nm.

In principle, there is no upper limit. However, for speed optimization of the screening method it is not very effective for the size of the miniaturized compartments to exceed several hundred micrometers, especially 500 micrometers. Preferable upper limit is 150 micrometers.

It is no longer necessary for individual compartments containing a sample to have a well shape, as known from conventional nanoscale titration plates, in order to optimize the speed in a holder miniaturized in this way. Suitable structures are, for example, grooves, cups or metallic bumps made by lithography and / or etching or stamping methods on silicon or other materials such as plastic or glass. It is also conceivable to be produced by a deposition structure or a self-assembly method. Microstructures made by the LIGA method can also be used.

For faster scanning of the compartment, it is sufficient for the compartment to have the form of a cup, trough or flat groove. The best design for multiple sample holders used in the new method or new device consists of a very flat planar disk on the sample to be irradiated, which is simply located in the form of droplets. If the substrate used is a Si wafer on a sample located in the form of droplets, it is preferable that the area in question is different from the surroundings in the surface structure. For example, if the wafer has regions coated with gold deposition, it is preferred that the individual compartments have a gold base, but hydrophobic silicon separates the individual compartments from each other.

The entire apparatus, including a single or multiple sample holder with a near microscope instrument, a compartment for a sample, and an adjustment mechanism, controls a microscope (already described above) in which the microscopy step is such that the adjustment instrument controls multiple sample holders rather than SNOM instruments. It can be adapted in a manner similar to moving in the area of light microscopy used.

The regulating mechanism itself may be similar to that known to those skilled in the art from other computer-controlled instruments, eg, stepping-motor regulating systems.

In general, the individual compartments of the multiple sample holders of this type can be arranged in any desired manner, in particular in rectangular or circular structure.

Examples of novel methods are shown below:

Fluorescence was detected using a commercially available SNOM instrument (eg Aurora, TopoMetrix Inc.). The substrate used was a gold-deposited ELISA plate or Si wafer. Thiolcarboxylic acid was applied to the substrate by liquid adsorption (self-assembly). The thiols bind to the gold surface and the acid is directed outwards. Relevant receptors such as avidin or thrombin were immobilized thereon via peptide bonds.

This was done by deriving the carboxyl-modified surface or tip with the spacers described above (eg aminohexanoic acid or spermidine) or other ligands. To this end, the carboxyl groups of the substrate or gold tip are 100 mM N-hydroxysuccinimide (C ₄ H ₅ NO ₃ ) and N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide (hydrochloride) for 5 minutes. Incubated with C ₈ H ₁₈ ClN ₃ and aminohexanoic acid or spermidine (100 mM each).

The aminohexanoic acid-derived surface is then reactivated with N-hydroxysuccinimide and N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide as described above and the amino-containing ligand (eg For example, thrombin or other protein) was reacted again at a concentration of about 100 μg / ml. However, it is also possible to bind low molecular weight ligands such as the following thrombin inhibitors to the surface.

If the spacer selected is spermidine, its free amino group can react with the activated carboxyl group of the ligand. This activated carboxyl group can likewise be produced by the aforementioned method. However, other known methods, such as peptide synthesis for activating carboxyl groups, are:

a) carboxylic acid is activated using chloroformic acid, EEDQ, IIDQ to obtain mixed anhydrides,

b) activating the carboxylic acid with carbodiimide to give a symmetric anhydride,

c) generally and possibly pentafluorophenols, hydroxysuccinimides, hydroxybenzotriazoles, using, for example, carbodiimides, urenium salts or urenium salts of benzotriazoles as active esters Or activating derivatives thereof,

d) As a halide or water halide, it can be used, for example, as fluoride using TFF, bromide using BrOP or azide.

Fluorescently-labeled avidin or thrombin was then added to directly bind the control active compound with information of the fluorescent complex on the surface of the substrate. These individual molecular fluorescences could be detected by SNOM. Then, when a mixture with a more effective active compound was added, labeled avidin or thrombin fell from its bond and dispersed from the surface with a decrease in fluorescence. However, the more effective active compounds present in the added mixture do not have to bind in the same place as the active compounds immobilized on the substrate surface. Reduction in binding of fluorescently-labeled molecules can also occur through binding of other sites on the substrate. Subsequent modification of the structure (an alternative site steric effect) reduces the affinity of the fluorescently-labeled molecules for the ligands immobilized on the substrate surface such that only a portion of the fluorescent molecules remain bound to the surface, thus reducing fluorescence .

Examples of cellular constructs that can be used as substrates are in particular membrane-fixed receptors, native signal-transducing receptors (e.g., receptors coupled to G-proteins, ligand- and voltage-dependent ion channels), or cells of the immune system. Mention may be made of phase receptors or platelets (eg, GPIIb / IIIa receptors) and pure binding proteins on the cells of major cellular targets.

Claims (23)

A method for screening an active compound, wherein the active compound is detected from a change in signal [S] that can be observed by a scanning near-field optical microscope instrument (SNOM instrument). A method according to claim 1, wherein the scanning optical near field is provided by an optical fiber (1). 3. The method of claim 1, wherein the active compound screening comprises providing multiple sample holders having multiple compartments for the sample, and all or part of such compartments, preferably by a parallel SNOM instrument. a) linking molecule A to substrate (2) covalently or noncovalently directly or indirectly, b) non-covalently binding molecule B to molecule A, which can be detected from signal [S] by scanning near-field microscopy, c) adding a solution containing active compound W2 to detect the presence of active compound W2 from the binding of molecule B and thus the change in signal [S]. The method of claim 1, wherein the active compound screening provides multiple sample holders having multiple compartments for a sample, and parallel or continuous to all or a portion of such compartments. a) to bind molecule A to substrate (2), b) adding a solution containing the active compound Wx, c) the active compound Wx is bound to A to form a molecular complex A-X, d) adding a solution containing molecular probe FITC that can be detected from signal [S] by scanning near-field microscopy, e) binding the molecular probe FITC to complex A-X, and f) detecting the binding of FITC to A-X by measuring [S]. The method according to any one of claims 1 to 4, wherein the signal [S] is a fluorescent signal. The method according to any one of claims 1 to 5, wherein the binding of molecule A to substrate (2) takes place by adsorption. The method according to any one of claims 1 to 6, wherein the substrate (2) is a Si wafer, a nano scale titration plate with a deposited Au coating, mica, graphite or cellular structure, or a combination thereof. 8. The method of claim 7, wherein the cellular construct is a membrane-anchored receptor. The method according to claim 1, wherein molecule A is indirectly bound to the substrate (2) via a spacer, in particular spermidine or aminohexanoic acid. 10. The process according to claim 9, wherein the spacers themselves are bound via a compound adsorbed on the substrate (2). The method according to claim 1, wherein a parallel measuring device comprising a SNOM instrument is used for simultaneous detection of multiple sample compartments for active compound screening. The method according to claim 1, wherein the signal [S] uses a difference in optical contrast, in particular adsorption, fluorescence, polarization or wavelength dependence, to detect binding of substance X. 12. The method according to any one of claims 10 to 12, wherein the spacer bonding step is repeated several times. Use of a scanning near optical microscope device (SNOM instrument) for screening active compound, wherein the active compound is detected from a change in signal [S] that can be observed by the scanning near optical microscope instrument. Use according to claim 14 for carrying out one of the methods of claims 1 to 13. A device comprising a near microscope device, a single or multiple sample holder having compartments for a sample, and an adjustment mechanism for addressing individual compartments of the single or multiple sample holders to the near microscope instrument. A device comprising a near microscope device, a single or multiple sample holder with compartments for a sample, and an adjustment mechanism for addressing the near microscope device by a single or multiple sample holder. 18. The device of any one of the preceding claims, wherein the single or multiple sample holders are multiwell nano scale titration plates. A multiple nanoscale titration device comprising a flat plate and a plurality of nanoscale compartments arranged thereon. 20. The multi-nanoscale titration device of claim 19 wherein the compartment size (diameter) is between 10 and 100 nm. The multi-nanoscale titration device of claim 19 or 20 wherein the nanoscale compartment is in the form of a well. The multi-nanoscale titration according to claim 19 or 20, wherein the nanoscale partition is bounded from the residual area in that the size (diameter) of the nanoscale partition is determined by a different chemical structure or composition than the residual area of the flat plate. device. 23. The multi-nanoscale titration device of claim 19, wherein the flat plate is hydrophobic silicon and the nanoscale partitions are defined by changes in surface structure.